Lyme disease is the leading tick-borne bacterial disease in the world resulting in greater than 30,000 cases per year in the US alone. Lyme disease is caused by tick-bite transmission of the pathogenic spirochete Borrelia burgdorferi. An increased understanding of the molecular mechanisms that B. burgdorferi uses to survive throughout its infectious cycle is critical for the development of innovative diagnostic and therapeutic protocols to reduce the incidence of Lyme disease. One of the major difficulties blocking this effort has been genome- wide identification of the B. burgdorferi genes that are expressed in the mammalian host environment. Using in vivo expression technology (IVET) in B. burgdorferi for the first time we have identified B. burgdorferi genes that are expressed during an active mammalian infection. Identification of in vivo-expressed spirochete genes will make a significant contribution toward elucidation of genetic mechanisms of B. burgdorferi gene regulation, host-pathogen interactions and persistence in the mammalian host. B. burgdorferi outer surface proteins are candidate virulence factors as they likely contribute to host-pathogen interactions as immune evasion proteins, adhesins and/or signaling molecules and may be novel serodiagnostic markers or vaccine candidates. Moreover, there is a growing understanding that nutrient acquisition functions are critical determinants of B. burgdorferi pathogenesis and may represent novel antimicrobial targets and/or a mechanism to deliver antimicrobial compounds to the pathogen. Therefore, characteristics of the four leading candidate genes from our IVET screen include: (i) encoded on a virulence associated plasmid, (ii) encode a putative outer surface protein and/or (iii) encode an uncharacterized transport system. This proposal is directed at understanding B. burgdorferi infection mechanisms through genetic, biochemical and in vivo analyses of unique in vivo- expressed genes. Discovery of novel B. burgdorferi virulence factors will advance our knowledge of Lyme disease pathogenesis and provide new diagnostic and antimicrobial targets for the improved treatment of this emerging infectious disease.